The genus Ganoderma, belonging to the family Ganodermataceae, comprises a group of basidiomycete fungi with wide geographic distribution and scientific interest. Despite its ecological relevance and biotechnological potential, its taxonomy remains complex due to high morphological variability among species, influenced by environmental and nutritional factors that directly affect phenotypic characteristics. This study aimed to perform morphological and molecular characterization of eight fungal isolates of Ganoderma to support taxonomic identification and provide insights into their phylogenetic relationships. The eight isolates evaluated were cultured in PDA medium, morphologically characterized, and subjected to microscopic observation of hyphal structures. Mycelial growth was evaluated in different media (MEA, PDA, and SDA) and temperatures (18°C, 28°C, and 38°C) to understand the isolates physiological behavior. Subsequently, genomic DNA extraction was performed, according to the protocol by Vicente et al. (2008) and sequencing of the ITS region of ribosomal DNA, used as a marker for molecular fungi identification. Microscopically, all strains presented the trimitic hyphal system, formed by generative, skeletal, and ligative hyphae. These structures, together with colony macromorphology, confirmed generic identity. However, species-level identification was only possible through molecular analysis, which revealed four strains of G. lucidum, one of G. sessile, one of G. lingzhi, and one with ambiguous identity between G. resinaceum and G. sessile. Physiological analysis showed that medium composition directly influenced development, with some combinations promoting mycelial expansion. The temperature of 28°C favored mycelial growth in most isolates, especially G. lucidum and G. lingzhi, indicating species-specific physiological profiles. These variations reveal the specific physiological profile of each species. These data show that the traditional approach, based on morphology, is limited in delimiting Ganoderma species. The integration of microscopic characteristics, and molecular data, is essential to ensure accurate identification.